Optimizing bioethanol production from sweet sorghum stem juice under very high gravity fermentation and temperature stress conditions

• Initial sugar, urea and cell concentrations impact ethanol fermentation from sweet sorghum stem juice under very high gravity condition. • Response surface methodology maximized ethanol concentration, productivity and sugar consumption. • Aeration enhanced cell viability but not fermentation efficiency under optimum conditions. • Osmoprotectant (KCl/KOH) improved ethanol fermentation efficiency at 25 °C and up to 37 °C. • Findings contribute to developing efficient bioethanol production under osmotic and temperature stresses. This study optimized ethanol production from sweet sorghum stem juice (SSJ) by Saccharomyces cerevisiae NP01 under very high gravity (VHG) fermentation in 500-mL air–locked flasks at 30 °C. Response surface methodology based on a Box-Behnken design was employed to optimize initial sugar (267 g/L), urea (3.24 g/L), and cell concentration (1.32 × 10 8 cells/mL) for maximization of ethanol concentration ( P E ), productivity ( Q P ), and sugar consumption ( %SC ). The experimental values ( P E , 119.29 g/L; Q P , 2.49 g/L.h and %SC , 91.83 %) under optimal conditions were close to the predicted values, verifying the optimization process. Aeration (2.5 vvm for 4 h) increased viable cell counts and decreased glycerol production (a by-product), but not fermentation efficiency. An osmoprotectant (40 mM potassium chloride combined with 10 mM potassium hydroxide, KCl/KOH) at 30 °C had no positive effect on ethanol fermentation efficiency. However, at 25 °C, the osmoprotectant increased P E from 106 to 116 g/L and ethanol yield from 0.46 to 0.49 g/g. At 35–37 °C, it prolonged cell viability, increasing P E by 5–12 g/L and %SC by 3–8 % without affecting ethanol yield. However, at 39 °C, no positive impact occurred on ethanol fermentation efficiency. The findings from this study, particularly the optimized fermentation conditions and stress tolerance strategies, could guide the scale-up to an industrial level of bioethanol production from sweet sorghum stem juice or other feedstocks using VHG fermentation, contributing to the development of more efficient and sustainable biofuel production processes.